LAY SUMMARY The efficacy of current hemostatic technologies is limited by several factors. Outward blood flow washes hemostatic drugs away from the wound, and hemostatic drugs often require focus, training, and time to use correctly, are highly specific to one type of injury, or pose severe safety risks. CounterFlow is a novel product that could potentially save military and civilian lives by stopping heavy bleeding from a variety of organs and other bodily locations that current technology cannot easily treat. Upon contact with blood, CounterFlow releases bursts of gas to safely self-propel bio-degradable clot-forming and clot-stabilizing drugs against blood flow, delivering them to the source of bleeding. This unique mechanism allows CounterFlow to be applied quickly to a wide assortment of wounds and to act effectively with little management after application. CounterFlow was tested in multiple animal models representing common and deadly bleeding scenarios, including internal bleeding, care under fire without compression, and surgical bleeding, and it was found to outperform current care options by stopping bleeds faster and increasing survival times. CounterFlow is also safe to use and biocompatible. This narrative review summarizes studies testing the effectiveness and safety of CounterFlow, discusses useful applications, and describes future plans for the product.

INTRODUCTION:Hemorrhage is responsible for 91% of preventable prehospital deaths in combat. Bleeding from anatomic junctions such as the groin, neck, and axillae make up 19% of these deaths, and reports estimate that effective control of junctional hemorrhage could have prevented 5% of fatalities in Afghanistan. Hemostatic dressings are effective but are time-consuming to apply and are limited when proper packing and manual pressure are not feasible, such as during care under fire. CounterFlow-Gauze is a hemostatic dressing that is effective without compression and delivers thrombin and tranexamic acid into wounds. Here, an advanced prototype of CounterFlow-Gauze, containing a range of low thrombin doses, was tested in a lethal swine model of junctional hemorrhage. Outcomes were compared with those of Combat Gauze, the current dressing recommended by Tactical Combat Casualty Care.MATERIALS AND METHODS:CounterFlow-Gauze containing thrombin doses of 0, 20, 200, and 500 IU was prepared. Swine received femoral arteriotomies, and CounterFlow-Gauze was packed into wounds without additional manual compression. In a separate study using a similar model of junctional hemorrhage without additional compression, CounterFlow-Gauze containing 500 IU thrombin was tested and compared with Combat Gauze. In both studies, the primary outcomes were survival to 3 h and volume of blood loss.RESULTS:CounterFlow-Gauze with 200 and 500 IU had the highest 3-h survival, achieving 70 and 75% survival, respectively. CounterFlow-Gauze resulted in mean peak plasma tranexamic acid concentrations of 9.6 ± 1.0 µg/mL (mean ± SEM) within 3 h. In a separate study with smaller injury, CounterFlow-Gauze with 500 IU achieved 100% survival to 3 h compared with 92% in Combat Gauze animals.CONCLUSIONS:An advanced preclinical prototype of CounterFlow-Gauze formulated with a minimized thrombin dose is highly effective at managing junctional hemorrhage without compression. These results demonstrate that CounterFlow-Gauze could be developed into a feasible alternative to Combat Gauze for hemorrhage control on the battlefield.

Collective cell migration is not only important for development and tissue homeostasis but can also promote cancer metastasis. To migrate collectively, cells need to coordinate cellular extensions and retractions, adhesion sites dynamics, and forces generation and transmission. Nevertheless, the regulatory mechanisms coordinating these processes remain elusive. Using A431 carcinoma cells, we identify the kinase MAP4K4 as a central regulator of collective migration. We show that MAP4K4 inactivation blocks the migration of clusters, whereas its overexpression decreases cluster cohesion. MAP4K4 regulates protrusion and retraction dynamics, remodels the actomyosin cytoskeleton, and controls the stability of both cell-cell and cell-substrate adhesion. MAP4K4 promotes focal adhesion disassembly through the phosphorylation of the actin and plasma membrane crosslinker moesin but disassembles adherens junctions through a moesin-independent mechanism. By analyzing traction and intercellular forces, we found that MAP4K4 loss of function leads to a tensional disequilibrium throughout the cell cluster, increasing the traction forces and the tension loading at the cell-cell adhesions. Together, our results indicate that MAP4K4 activity is a key regulator of biomechanical forces at adhesion sites, promoting collective migration.

ABSTRACT Collective cell migration is important for normal development and tissue homeostasis, but can also promote cancer metastasis. To migrate collectively, cells need to coordinate their protrusion formation, rear retraction, adhesion sites dynamics, as well as forces generation and transmission. Nevertheless, the regulatory mechanisms coordinating these processes remain elusive. Using the A431 carcinoma cell line, we identify the kinase MAP4K4 as a central regulator of collective migration. We show that MAP4K4 inactivation blocks the migration of clusters while its overexpression decreases cluster cohesion. MAP4K4 regulates protrusion and retraction dynamics, remodels the actomyosin cytoskeleton, and controls the stability of both cell-cell and cell substrate adhesion. MAP4K4 promotes focal adhesion disassembly through the phosphorylation of Moesin, an actin and plasma membrane cross-linker, but disassembles adherens junctions through a Moesin-independent mechanism. By analyzing traction and intercellular forces, we found that the stabilization of adhesion sites in MAP4K4 loss of function leads to a tensional disequilibrium throughout the cell cluster, increasing the traction forces exerted onto the substrate and the tension loading at the cell-cell adhesions. Together, our results indicates that MAP4K4 activity is a key regulator of biomechanical forces at adhesion sites, promoting collective migration.

Tissue and cell mechanics are crucial factors in maintaining homeostasis and in development, with aberrant mechanics contributing to many diseases. During the epithelial-to-mesenchymal transition (EMT), a highly conserved cellular program in organismal development and cancer metastasis, cells gain the ability to detach from their original location and autonomously migrate. While a great deal of biochemical and biophysical changes at the single-cell level have been revealed, how the physical properties of multicellular assemblies change during EMT, and how this may affect disease progression, is unknown. Here we introduce cell monolayer deformation microscopy (CMDM), a new methodology to measure the planar mechanical properties of cell monolayers by locally applying strain and measuring their resistance to deformation. We employ this new method to characterize epithelial multicellular mechanics at early and late stages of EMT, finding the epithelial monolayers to be relatively compliant, ductile, and mechanically homogeneous. By comparison, the transformed mesenchymal monolayers, while much stiffer, were also more brittle, mechanically heterogeneous, displayed more viscoelastic creep, and showed sharp yield points at significantly lower strains. Here, CMDM measurements identify specific biophysical functional states of EMT and offer insight into how cell aggregates fragment under mechanical stress. This mechanical fingerprinting of multicellular assemblies using new quantitative metrics may also offer new diagnostic applications in healthcare to characterize multicellular mechanical changes in disease.

IntroductionNon-compressible intra-abdominal hemorrhage (NCIAH) is a major cause of preventable death on the battlefield and in civilian trauma. Currently, it can only be definitively managed with surgery, as there are limited strategies for controlling ongoing NCIAH in the prehospital environment. We hypothesized that a self-propelling thrombin-containing powder (SPTP) could increase survival in a swine model of NCIAH when delivered percutaneously into the closed abdomen using an engineered spray system.Materials and MethodsNineteen swine underwent surgical laparotomy followed by a Grade V liver injury that created massive hemorrhage, before closing the abdomen with sutures. Animals either received treatment with standard of care fluid resuscitation (n=9) or the SPTP spray system (n=10), which consisted of a spray device and a 14 Fr catheter. Using the spray system, SPTP was delivered into a hemoperitoneum identified using a focused assessment with sonography in trauma (FAST) exam. Lactated Ringer's solution was administered to all animals to maintain a mean arterial pressure (MAP) of >50 mmHg. The primary outcome was percentage of animals surviving at three hours following injury.ResultsIn the swine model of NCIAH, a greater percentage of animals receiving SPTP survived to three hours, although differences were not significant. The SPTP spray system increased the median survival of animals from 1.6 hr in the fluid resuscitation group to 4.3 hr. The SPTP spray system delivered a total mass of 18.5 ± 1.0 g of SPTP. The mean change in intra-abdominal pressure following SPTP delivery was 5.2 ± 1.8 mmHg (mean ± SEM). The intervention time was 6.7 ± 1.7 min. No adverse effects related to the SPTP formulation or the spray system were observed. SPTP was especially beneficial in animals that had either severely elevated lactate concentrations or low mean arterial pressure of <35 mmHg shortly after injury.ConclusionsThis demonstrates proof-of-concept for use of a new minimally invasive procedure for managing NCIAH, which could extend survival time to enable patients to reach definitive surgical care.

Glasses have numerous applications because of their exceptional transparency and stiffness; however, poor fracture, impact resistance, and mechanical reliability limit the range of their applications. Recent bioinspired glasses have shown superior mechanical performance, but they still suffer from reduced optical quality. Here, we present a nacreous glass composite that offers a combination of strength, toughness, and transparency. Micrometer-sized glass tablets and poly(methyl methacrylate) (PMMA) were mixed and structured by centrifugation, creating dense PMMA-glass layers. A transparent composite was created by tuning the refractive index of PMMA to that of glass and using chemical functionalization to create continuous interfaces. The fabrication method is robust and scalable, and the composite may prove to be a glass alternative in diverse applications.

Traction Force Microscopy (TFM) is broadly used to measure cell contractility and its role in cell behavior. While TFM platforms have enabled diverse discoveries, their implementation remains limited in part due to various methodology and imaging constraints. Here we introduce a reference-free technique to measure cell contractile work in real-time, with simple substrate fabrication, imaging, and analysis with the availability of the cells for post-processing. in this technique, we confine the cells on fluorescent adhesive protein micropatterns of a known area on compliant silicone substrates (2,12 and 23 kPa) and use the cell deformed pattern area to calculate cell contractile work (Strain Energy). We validated this approach by comparing contractile work of 3T3 Fibroblast cells measured with PaCS to conventional bead-displacement TFM and show quantitative agreement between the methodologies. Using this platform, we show PaCS accurately detects cell contractile work across diverse cell shapes and substrate stiffness. It detects time-dependent contractile changes in cells and exhibits a decrease in cell contractility after treatment in inhibitors. PaCS precisely differentiates the higher contractile work done by highly metastatic MDA-MB-231 breast cancer cells when compared with MCF-7 cells with lower metastatic potential. The ability of PaCS to measure contractile work just from the pattern deformations fills a current void in simplified cell contractile methodologies and provides a promising future for the incorporation of cell biophysics in modern quantitative biology studies.

The sensing and generation of cellular forces are essential aspects of life. Traction force microscopy (TFM) has emerged as a standard broadly applicable methodology to measure cell contractility and its role in cell behavior. While TFM platforms have enabled diverse discoveries, their implementation remains limited in part due to various constraints, such as time-consuming substrate fabrication techniques, the need to detach cells to measure null force images, followed by complex imaging and analysis, and the unavailability of cells for postprocessing. Here we introduce a reference-free technique to measure cell contractile work in real time, with commonly available substrate fabrication methodologies, simple imaging, and analysis with the availability of the cells for postprocessing. In this technique, we confine the cells on fluorescent adhesive protein micropatterns of a known area on compliant silicone substrates and use the cell deformed pattern area to calculate cell contractile work. We validated this approach by comparing this pattern-based contractility screening (PaCS) with conventional bead-displacement TFM and show quantitative agreement between the methodologies. Using this platform, we measure the contractile work of highly metastatic MDA-MB-231 breast cancer cells that is significantly higher than the contractile work of noninvasive MCF-7 cells. PaCS enables the broader implementation of contractile work measurements in diverse quantitative biology and biomedical applications.

The mechanics of multicellular clusters are essential for processes such as collective motion, supracellular mechano-sensing, and diverse changes in physiology and pathology. One example of multicellular pathology is the detachment of metastatic cells from their original network, whereupon they become migratory. The epithelial-mesenchymal transition (EMT) is one well known process whereby malignant cells can fragment from each other and become invasive. However, the changes in the collective intercellular mechanics of cells as they progress through EMT are unknown. Here we present a new methodology, Cell Monolayer Deformation Microscopy (CMDM), to locally strain cell monolayers and measure the monolayer's planar rheology. We use image cross-correlation algorithms to determine the differences in poly-N-isopropylacrylamide (PNIPAm) hydrogel substrate deformation with and without an attached cell monolayer. We then quantify the resistance of the cell monolayers to lateral stretching, and calculate deformability, yielding, and time-dependent viscoelastic creep-/relaxation-associated deformation. We applied this technique to examine changes in the mechanical behavior of NMuMG cell monolayers in their epithelial phenotype, and in their Transforming Growth Factor-ß (TGFß) induced mesenchymal phenotype, to capture the differences in collective cell mechanics during EMT. We found that the epithelial cell monolayers are softer, whilst also undergoing less time-dependent creep under applied strain. interestingly, yielding in the epithelial monolayer appears more stress-dependent than strain-dependent. Mesenchymal monolayer yielding is highly strain-dependent, and they exhibit mechanical yielding at lower strains than the epithelial monolayers. This CMDM methodology is further applicable to other 2D cell monolayer systems, and will help to resolve the complex mechanics of multicellular structures.

Glasses have numerous applications due to their exceptional transparency, however, poor fracture and impact resistance limit their applications as an engineering material. One relatively recent approach to improve the mechanical properties of materials is through bio-inspiration. Structural biological composites such as nacre, the protective inner layer of mollusk shells, offer far superior mechanical properties relative to their constituents. This has motivated researchers to mimic the design principles in natural composites to create tough transparent materials. However, current bio-inspired materials lack fabrication scalability or offer poor optical transmission. Here, an efficient, scalable bulk process is developed for creating optically transparent tough composites, resulting in a nacreous glass composite material with a four-fold increase in fracture toughness and a three-fold increase in flexural strength compared to conventional structural glasses, and with a 73% of average optical transmittance. The composite consists of glass flakes and poly (methyl methacrylate) (PMMA) assembled utilizing a centrifuge-based fabrication method that aligns and compacts the flakes into layers. To optimize the transparency of the structure, the refractive indices of the PMMA and glass are matched. Based on the results, this nacreous glass composite is proposed as a potential alternative in diverse architectural, vehicular, and electronics applications.

Measuring pressures within complex multi-cellular environments is critical for studying mechanobiology as these forces trigger diverse biological responses, however, these studies are difficult as a deeply embedded yet well-calibrated probe is required. In this manuscript, we use endogenous cell nuclei as pressure sensors by introducing a fluorescent protein localized to the nucleus and confocal microscopy to measure the individual nuclear volumes in 3D multi-cellular aggregates. We calibrate this measurement of nuclear volume to pressure by quantifying the nuclear volume change as a function of osmotic pressure in isolated 2D culture. Using this technique, we find that in multicellular structures, the nuclear compressive mechanical stresses are on the order of MPa, increase with cell number in the cluster, and that the distribution of stresses is homogenous in spherical cell clusters, but highly asymmetric in oblong clusters. This approach may facilitate quantitative mechanical measurements in complex and extended biological structures both in vitro and in vivo.

The actin cytoskeleton is known to be a main structural and mechanical component of many eukaryotic cells. Dynamic crosslinking of actin filaments by proteins such as alpha-actinin, can change these mechanics, however, the effect of dynamic crosslinking on cell mechanics has not been previously reported. Here we quantify the viscoelastic moduli of cells with alpha-actinin isoforms that have varied binding affinity. We apply several techniques, including passive and active micro-rheology and AFM measurements to measure cellular moduli with alpha-actinin isoforms. The experiments are also performed after myosin inhibition and ATP depletion to study the contribution of active contractility to the viscoelastic properties of these cells. AFM-based stress relaxation and dynamic indentation tests are used to calculate the mechanical properties of the cellular cortex, while active micro-rheology using optical tweezers allows us to calculate the storage and loss moduli of the cytoplasm. Furthermore, the overall energy dissipation is estimated at cellular level, and these results are compared with cell work on the elastic substrates, using traction force microscopy data. Energy dissipation via dynamic crosslinkers in the cytoskeleton can regulate cellular viscoelasticity and basic biological functions such as spreading, force-generation, and migration.

Event Abstract Back to Event Tunable viscoelastic polydimethylsiloxane substrates for cell mechanics and mechanobiology applications Hossein Heris1, Adele Khavari1, 2 and Allen J. Ehrlicher1 1 McGill University, Bioengineering, Canada 2 Chalmers University of Technology, Chemistry and Chemical Engineering, Sweden Introduction: The physical and mechanical properties of the extracellular matrix (ECM) are known to influence and regulate cell fate and a wide variety of biological processes such as cell migration and differentiation. However, the underlying physical mechanisms behind the interactions between cells and their microenvironment is not well understood. Many studies have revealed profound effects of substrate elasticity on cellular responses. For instance, it has been shown that morphology, cytoskeletal structure, and cellular adhesion change in response to substrate elasticity[1]. Substrate elasticity has also been shown to guide cell migration[2] and to direct stem cell differentiation[3]. In most of these studies, purely elastic materials were used to characterize cell-biomaterial interactions, however, most biological materials are viscoelastic and exhibit time-dependent deformation to the applied force, usually referred to as creep behavior. One clear consequence of the material’s creep is energy dissipation. Unlike purely elastic materials where elastic energy is conserved, viscoelastic materials dissipate energy and constantly require input work to maintain a constant stress, commensurate with the rate of energy dissipation. The goal of this work is to establish a new platform to study mechanobiology and cell mechanics in response to viscoelastic properties of the ECM. Material and Method: Polydimethylsiloxane (PDMS) was purchased from Gelest. PDMS was functionalized for cell adhesion. A rheometer was used to characterize the viscoelastic properties of the substrates. Parallel plates with a diameter of 20 mm and a gap of 1000 µm were used. Frequency sweep and creep tests were performed. NIH 3T3 fibroblasts cells were cultured in a mixture of Dulbecco’s Modified Eagle Medium, 10% fetal bovine serum, 1% Penicillin/Streptomycin at 37ºC, in 5% CO2 humidified atmosphere. Cells were disassociated using 0.25% trypsin-EDTA when the cell confluency reached 70% and were cultured on the PDMS substrates' surface. Results: The fabricated PDMS substrates exhibit tunable viscoelastic properties. The shear and loss moduli were tunable between 4- 50 kPa and 0.2-7 kPa, respectively. Figure 1 shows the creep response of three PDMS samples. The shear stress was 2 kPa during creep test. This result indicates constant shear moduli in long time scale for these three samples but different time scale for their creep behavior. Cell culture results showed the spreading of NIH 3T3 cells on PDMS substrates. Discussion: Tunable viscoelastic PDMS substrates can provide us with a very strong tool to understand how cells sense and respond to the mechanical properties of their surrounding matrix. This viscoelastic platform can facilitate quantitative characterization of cell-ECM interactions to understand cell behavior during development and pathological conditions. Conclusions: PDMS-based tunable viscoelastic substrates with cell adhesion functionalities were fabricated and characterized for use in studying cell mechanics and mechanobiology. Canada Foundation for Innovation; Canadian Institute of Health Research; Natural Sciences and Engineering Research Council of CanadaReferences:[1] Yeung T et al, “Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion”. Cell Motil Cytoskeleton. 2005; 60, 24-34.[2] Lo CM et al, “Cell Movement Is Guided by the Rigidity of the Substrate”, Biophysical journal, 2000, 79, 144-152.[3] Angler AJ et al, “Matrix elasticity directs stem cell lineage specification”, Cell. 2006, 126: 677-89. Keywords: Cell Adhesion, Cell response, mechanical property, matrix-cell interaction Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Mechanobiology of cells on biomaterials Citation: Heris H, Khavari A and Ehrlicher AJ (2016). Tunable viscoelastic polydimethylsiloxane substrates for cell mechanics and mechanobiology applications. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.02958 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Hossein Heris Adele Khavari Allen J Ehrlicher Google Hossein Heris Adele Khavari Allen J Ehrlicher Google Scholar Hossein Heris Adele Khavari Allen J Ehrlicher PubMed Hossein Heris Adele Khavari Allen J Ehrlicher Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

The mechanics of the cellular microenvironment can be as critical as biochemistry in directing cell behavior. Many commonly utilized materials derived from extra-cellular-matrix create excellent scaffolds for cell growth, however, evaluating the relative mechanical and biochemical effects independently in 3D environments has been difficult in frequently used biopolymer matrices. Here we present 3D sodium alginate hydrogel microenvironments over a physiological range of stiffness (E = 1.85 to 5.29 kPa), with and without RGD binding sites or collagen fibers. We use confocal microscopy to measure the growth of multi-cellular aggregates (MCAs), of increasing metastatic potential in different elastic moduli of hydrogels, with and without binding factors. We find that the hydrogel stiffness regulates the growth and morphology of these cell clusters; MCAs grow larger and faster in the more rigid environments similar to cancerous breast tissue (E = 4–12 kPa) as compared to healthy tissue (E = 0.4–2 kpa). Adding binding factors from collagen and RGD peptides increases growth rates, and change maximum MCA sizes. These findings demonstrate the utility of these independently tunable mechanical/biochemistry gels, and that mechanical confinement in stiffer microenvironments may increase cell proliferation.

The sol-to-gel transition of an alginate rich in β-d-mannuronic acid residues and at a concentration of 0.1% w/v in 15 mM NaCl in the presence of calcium ions of 0 to 3.5mM was studied with dynamic light scattering. The dynamics of the different systems added further insight into the alginate gel forming mechanisms. Below a Ca(2+) concentration of 0.7 mM, the build-up of small aggregates could be verified. Moreover, at a critical concentration, close to 0.9 mM Ca(2+), a percolated, non-ergodic network started to form from some of these aggregates, with smaller aggregates still diffusing in the network. The system displayed strong non-ergodicy at high Ca(2+) concentrations with a non-ergodicity parameter that appeared to form discontinuously from near zero to a clearly non-zero value at the critical Ca(2+) concentration.